A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

Background

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results

We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions

Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.     

7-nitroindazole and posthypoxic ventilatory behavior in the A/J and C57BL/6J mouse strains.

Periodic breathing (PB) is a fundamental breathing pattern in many common cardiopulmonary illnesses. The finding of PB in C57BL/6J (B6) mice was previously ascribed to strain differences in posthypoxic ventilatory and frequency decline in the B6 mice (Han F, Subramanian S, Price ER, Nadeau J, and Strohl KP. J Appl Physiol 92: 1133-1140, 2002). We tested whether the induction of posthypoxic frequency decline in A/J mice, through administration of a neuronal nitric oxide synthase blocker [7-nitroindazole (7-NI); 60 mg/kg], would cause A/J mice to exhibit PB and/or alter PB expression in the B6 strain. Recordings of ventilatory behavior by the plethysmography method were made when unanesthetized B6 (n = 10) or A/J (n = 6) animals were reoxygenated with 100% O2 or room air after exposure to 8% O2. Before undergoing gas challenges, mice were given an intraperitoneal injection of either peanut oil alone (vehicle) or 7-NI suspended in peanut oil. Compared with vehicle, both strains of mice exhibited posthypoxic frequency decline and the absence of short-term potentiation with 7-NI administration. B6 mice continued to exhibit posthypoxic PB; however, the PB was characterized by longer cycle and apnea length. In contrast, A/J mice did not show increased tendency toward posthypoxic PB with 7-NI. We conclude that 7-NI further differentiates the A/J and B6 strains in terms of PB and that strain-related differences in posthypoxic frequency decline are not primary determinants of this strain difference in the occurrence of PB. Metabolism was not associated with either the expression of posthypoxic ventilatory decline or PB. Furthermore, neuronal nitric oxide may be an organizing feature in the presence, length, and/or cycle length of apnea in the susceptible strain.     

Genetic polymorphisms among C57BL/6 mouse inbred strains

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina^® Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyr ^c?2j /J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.     

Effects of buspirone on posthypoxic ventilatory behavior in the C57BL/6J and A/J mouse strains.

Buspirone, a partial agonist of the serotonergic 5-HT1A receptor, improves breathing irregularities in humans with Rett syndrome or brain stem injury. The purpose of this study was to examine whether buspirone alters posthypoxic ventilatory behavior in C57BL/6J (B6) and A/J mouse strains. Measurements of ventilatory behavior were collected from unanesthetized adult male mice (n=6 for each strain) using the plethysmographic method. Mice were given intraperitoneal injections of vehicle or several doses of buspirone and exposed to 2 min of hypoxia (10% O2) followed by rapid reoxygenation (100% O2). Twenty minutes later, mice were tested for hypercapnic response (8% CO(2)-92% O2). On a separate day, mice were injected with the 5-HT1A receptor antagonist 4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl] ethyl}-N-2-pyridinylbenzamide (p-MPPI) before the injection of buspirone, and measurements were repeated. In separate studies, arterial blood-gas analysis was performed for each strain (n=12 in B6 and 10 in A/J) with buspirone or vehicle. In both strains, buspirone stimulated ventilation at rest. In the B6 mice, the hypoxic response was unchanged, but the response to hypercapnia was reduced with buspirone (5 mg/kg; P<0.05). With reoxygenation, vehicle-treated B6 exhibited periodic breathing and greater variation in ventilation compared with A/J (P<0.01). In B6 animals, >or=3 mg/kg of buspirone reduced variation and prevented the occurrence of posthypoxic periodic breathing. Both effects were reversed by p-MPPI. Treatment effect of buspirone was not explained by a difference in resting arterial blood gases. We conclude that buspirone improves posthypoxic ventilatory irregularities in the B6 mouse through its agonist effects on the 5-HT1A receptor.     

Nuclear proteome profile of C57BL/6J mouse liver

The liver proteome can serve as a reference to better understand both disease mechanisms and possible therapeutics,since the liver is an important organ in the body that performs a large number of tasks.Here we identify the organelle proteome of C57BL/6J mouse liver nuclei as a promising strategy to enrich low abundance proteins,in the sense that analysis of whole liver cells is rather complex for current techniques and may not be suitable for proteins with low abundance.Evaluation of nucleus integrity and purity was performed to demonstrate the effectiveness of the optimized isolation procedure.The extracted nuclear proteins were identified by 2-DE MS analyses,and a total of 748 proteins were identified.Bioinformatic analyses were performed to demonstrate the physicochemical properties,cellular locations and functions of the proteins.     

A genome-wide set of congenic mouse strains derived from DBA/2J on a C57BL/6J background

In the analysis of complex traits, congenic strains are powerful tools because they allow characterization of a single locus in the absence of genetic variation throughout the remainder of the genome. Here, we report the construction and initial characterization of a genome-wide panel of congenic strains derived from the donor strain DBA/2J on the background strain C57BL/6J. For many strains, we have carried out high-density SNP genotyping to precisely map the congenic interval and to identify any contaminating regions. Certain strains exhibit striking variation in litter size and in the ratio of females to males. We illustrate the utility of the set by "Mendelizing" the complex trait of myocardial calcification. These 65 strains cover more than 95% of the autosomal genome and should facilitate the analysis of the many genetic trait differences that have been reported between these parental strains.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Quantitative trait loci analysis for the differences in susceptibility to atherosclerosis and diabetes between inbred mouse strains C57BL/6J and C57BLKS/J.

Mice from the inbred strain C57BLKS/J (BKS) exhibit increased susceptibility to both diabetes and atherosclerosis compared to C57BL/6J (B6) mice. To determine whether the differences in diabetes and atherosclerosis are related, we carried out a cross between B6-db/db and BKS. We selected 99 female F2-db/db progeny, tested the progeny for plasma lipids, plasma glucose, and fatty-streak lesions, and used quantitative trait loci (QTL) analysis to identify the chromosomal regions associated with these phenotypes. No major QTL were found for total cholesterol, VLDL-cholesterol, or triglycerides. Two suggestive QTL were found for HDL-cholesterol (LOD scores of 2. 7 and 2.8), and two suggestive loci were found for plasma glucose (LOD scores of 2.3 and 2.0). Lesion size was not correlated with plasma lipid levels or glucose. Lesion size was determined by a locus at D12Mit49 with a LOD score of 2.5 and a significant likelihood ratio statistic. The gene for apolipoprotein apoB lies within the region, but apoB levels were similar in strains B6 and BKS. The QTL on Chr 12 was confirmed by constructing a congenic strain with BKS alleles in the QTL region on a B6 genetic background. We conclude that susceptibilities to diabetes and atherosclerosis are not conferred by the same genes in these strains and that a major gene on Chr 12, which we name Ath6, determines the difference in atherosclerosis susceptibility.     

Pavlovian biconditional discrimination learning in the C57BL/6J mouse

Four experiments using mice examined acquisition of Pavlovian biconditional discriminations in which two stimulus compounds were paired with food (AX+ and BY+) and two were not (AY- and BX-). Temporally asynchronous compounds were generated by using contextual stimuli (Experiment 1) and 15-s discrete visual cues (Experiments 2A, 2B and 3) to disambiguate when embedded noise or tone stimuli would be paired with food. When food pellets followed both reinforced compounds, successful acquisition was obtained in Experiment 1 but not in Experiments 2A and 2B even though the order of trials was modeled after that used in Experiment 1. However, when differential outcomes followed the reinforced compounds in Experiment 3, acquisition was obtained with discrete cue stimulus compounds. The implications of these results for modulatory models of conditional discrimination learning in animals are discussed.     

Genetic differentiation within the inbred C57BL/6J mouse strain

The C57BL/6J (B6) inbred mouse strain is commonly used in biomedical researches. However, some unexpected inconsistency was reported compared with previous studies, and in most cases, it can be attributed to environmental, epigenetic or stochastic differences. The goal of this study was to investigate the genetic stability of the B6 strain maintained in different breeders. B6 mice purchased from five Chinese commercial breeders were examined, and mitochondrial D-loop sequence and 18 microsatellite loci were genotyped. There is no difference in the D-loop sequences, but variations exist in the nucleic microsatellite markers. Combining the data from MGI_4.01, a significant divergence is observed among those mice. The present study indicates that different B6 mice share the common maternal lineage and are still inbred in each breeder, but subline divergence occurs.     

Differences in anxiety-related behavior between apolipoprotein E-deficient C57BL/6 and wild type C57BL/6 mice

The influence of ApoE gene deletion on the anxiety state has not been previously investigated. The elevated plus maze was used in this study to determine differences in anxiety-related behavior between apoE-deficient and wild type C57BL/6 mice. The apoE-deficient mice demonstrated less anxiety on the elevated plus maze by spending more time in the open arms of the elevated plus maze compared to wild type mice (p<0.001). Additionally, female apoE-deficient mice visited the open arm of the maze more often than their apoE-deficient male counterpart (p<0.05). The anxiety state and/or sex are possible variables to be considered when designing physiological and/or behavioral studies involving mice that are apoE-deficient.     

Acetazolamide protects against posthypoxic unstable breathing in the C57BL/6J mouse.

Acetazolamide (Acz), a carbonic anhydrase inhibitor, is used to manage periodic breathing associated with altitude and with heart failure. We examined whether Acz would alter posthypoxic ventilatory behavior in the C57BL/6J (B6) mouse model of recurrent central apnea. Experiments were performed with unanesthetized, awake adult male B6 mice (n = 9), ventilatory behavior was measured using flow-through whole body plethysmography. Mice were given an intraperitoneal injection of either vehicle or Acz (40 mg/kg), and 1 h later they were exposed to 1 min of 8% O(2)-balance N(2) (poikilocapnic hypoxia) or 12% O(2)-3% CO(2)-balance N(2) (isocapnic hypoxia) followed by rapid reoxygenation (100% O(2)). Hypercapnic response (8% CO(2)-balance O(2)) was examined in six mice. With Acz, ventilation, including respiratory frequency, tidal volume, and minute ventilation, in room air was significantly higher and hyperoxic hypercapnic ventilatory responsiveness was generally lower compared with vehicle. Poikilocapnic and isocapnic hypoxic ventilatory responsiveness were similar among treatments. One minute after reoxygenation, animals given Acz exhibited posthypoxic frequency decline, a lower coefficient of variability for frequency, and no tendency toward periodic breathing, compared with vehicle treatment. We conclude that Acz improves unstable breathing in the B6 model, without altering hypoxic response or producing short-term potentiation, but with some blunting of hypercapnic responsiveness.     

Adrenal function and the effect of a high-fat diet on C57BL/6J and C57BL/6J-ob/ob mice.

In C57BL/6J mice and the ob/+ and ob/ob mutants total plasma corticosterone levels were found to be statistically different. In C57BL/6J mice the level was 1.9 +/- 0.2 mug/100 ml plasma, in ob/+ mice 8.6 +/- 1.6 mug/100 ml and in ob/ob mice 13.7 +/- 1.5 mug/100 ml. The percentage of protein-bound corticosterone as well as the free endogenous corticosterone levels were also different. Feeding a high-fat diet to young C57BL/6J and C57BL/6J-ob/ob mice for a period of 4 weeks had no effect upon blood glucose, plasma insulin and plasma corticosterone levels. The significantly higher increase in body weight of the high-fat diet groups of both lines of mice was mainly due to fat cell hypertrophy.     

C57BL/6J and B6.V-LEPOB mice differ in the cholinergic modulation of sleep and breathing.

Respiratory and arousal state control are heritable traits in mice. B6.V-Lep(ob) (ob) mice are leptin deficient and differ from C57BL/6J (B6) mice by a variation in the gene coding for leptin. The ob mouse has morbid obesity and disordered breathing that is homologous to breathing of obese humans. This study tested the hypothesis that microinjecting neostigmine into the pontine reticular nucleus, oral part (PnO), of B6 and ob mice alters sleep and breathing. In B6 and ob mice, neostigmine caused a concentration-dependent increase (P < 0.0001) in percentage of time spent in a rapid eye movement (REM) sleeplike state (REM-Neo). Relative to saline (control), higher concentrations of neostigmine increased REM-Neo duration and the number of REM-Neo episodes in B6 and ob mice and decreased percent wake, percent non-REM, and latency to onset of REM-Neo (P < 0.001). In B6 and ob mice, REM sleep enhancement by neostigmine was blocked by atropine. Differences in control amounts of sleep and wakefulness between B6 and the congenic ob mice also were identified. After PnO injection of saline, ob mice spent significantly (P < 0.05) more time awake and less time in non-REM sleep. B6 mice displayed more (P < 0.01) baseline locomotor activity than ob mice, and PnO neostigmine decreased locomotion (P < 0.0001) in B6 and ob mice. Whole body plethysmography showed that PnO neostigmine depressed breathing (P < 0.001) in B6 and ob mice and caused greater respiratory depression in B6 than ob mice (P < 0.05). Western blot analysis identified greater (P < 0.05) expression of M2 muscarinic receptor protein in ob than B6 mice for cortex, midbrain, cerebellum, and pons, but not medulla. Considered together, these data provide the first evidence that pontine cholinergic control of sleep and breathing varies between mice known to differ by a spontaneous mutation in the gene coding for leptin.     

Hippocampal lipids linked to spatial memory in the C57BL/6J mouse

Although the role of individual brain lipids for learning and memory has been reported, no systematic approach associating brain lipids with spatial memory has been carried out. It was therefore the aim of the study to determine brain lipids in hippocampus of mice forming and yoked controls that did not form spatial memory using the probe trial as the endpoint. 10 animals were trained in the Morris water maze (MWM) and 10 mice were serving as yoked controls i.e. no platform was used during the whole experiment. Hippocampal tissue lipids were extracted and data were acquired with Fourier transformation ion cyclotron resonance mass spectrometry (LTQ-FT) coupled to HPLC. Glycerophosphatidylethanolamines (18:0/22:6, 18:0/20:4 and 18:1/18:1), plasmalogens (16:0-10/22:6 and 18:0-10/22:6) and ceramides (18:0) showed higher levels in the trained group, while glycerolysophosphatidylcholines (16:0, 18:1, 18:0, 20:4), sphingomyelins (16:0, 24:1), ether linked glycerophosphatidylcholines (16:0-10/18:0), glycerophosphatidylcholines (16:0/18:1, 16:0/18:0, 18:0/18:1, 38:7, 18:1/20:1, 20:4/20:4, 22:1/18:1, 22:0/18:1, 20:4/22:6, 22:6/22:6), glucosylceramide (24:1) and plasmalogen (18:0-10/20:1) revealed lower levels in the trained group. Decreased levels of certain species of lysophosphatidylcholine, sphingomyelin, plasmenylphosphatidylcholine, phosphatidylcholine, glycosylceramide and plasmalogen at the probe trial for spatial memory may indicate catabolism in terms of consumption during this process. Increased hippocampal levels of long chain highly unsaturated phosphatidylethanolamines, plasmalogens and ceramides may reflect increased synthesis or decreased degradation at the endpoint of memory testing, probably representing interactions in the brain lipid pathways. The study shows pathways involved in spatial memory, may propose the use of individual brain lipids as probable cognitive enhancers and forms the basis for further studies on the role of brain lipids per se.     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.     

Differential regulation of leptin expression and function in A/J vs. C57BL/6J mice during diet-induced obesity

Obesity-resistant (A/J) and obesity-prone (C57BL/6J) mice were weaned onto low-fat (LF) or high-fat (HF) diets and studied after 2, 10, and 16 wk. Despite consuming the same amount of food, A/J mice on the HF diet deposited less carcass lipid and gained less weight than C57BL/6J mice over the course of the study. Leptin mRNA was increased in white adipose tissue (WAT) in both strains on the HF diet but to significantly higher levels in A/J compared with C57BL/6J mice. Uncoupling protein 1 (UCP1) and UCP2 mRNA were induced by the HF diet in brown adipose tissue (BAT) and WAT of A/J mice, respectively, but not in C57BL/6J mice. UCP1 mRNA was also significantly higher in retroperitoneal WAT of A/J compared with C57BL/6J mice. The ability of A/J mice to resist diet-induced obesity is associated with a strain-specific increase in leptin, UCP1, and UCP2 expression in adipose tissue. The findings indicate that the HF diet does not compromise leptin-dependent regulation of adipocyte gene expression in A/J mice and suggest that maintenance of leptin responsiveness confers resistance to diet-induced obesity.